The invention relates to the extraction of RNA for use in, among other things, in vitro diagnostics.
As a result of recent progress in genomic technology and bioinformatics, new gene expression markers useful as diagnostic, prognostic and therapeutic indicators for cancer and other diseases are rapidly being discovered. To evaluate and apply these markers as in vitro diagnostic tests, there is a need for a rapid, sensitive, and easy-to-use method of RNA extraction from cells, tissues, and other biological components.
In oncology, rapid intra-operative molecular testing may be used to more sensitively detect surgical margins, lymph node or other sites of metastases, and to confirm the presence or absence of cancer in a tissue. To impact surgical care, diagnostic results useful in the intraoperative setting must be made available to the treating surgeon during the time that the patient is in the operating room, generally about 45 min. To allow time for reverse transcription and subsequent amplification and detection of molecular markers, amplifiable RNA from cells and tissues must be extracted and purified within a few minutes.
Several different approaches have been developed for the isolation and purification of RNA from cells and tissues. These include membrane-based methods, solution based methods, and magnetic based methods. Solution based methods generally require at least 90 minutes to perform and involve the use of toxic solvents. Magnetic particle methods have been developed but require at least 45 min to perform.
Kits for performing membrane based RNA purification are commercially available from several vendors. Generally, kits are-developed for the small-scale (30 mg or less) preparation of RNA from cells and tissues (eg. QIAGEN RNeasy Mini kit), for the medium scale (250 mg tissue) (eg. QIAGEN RNeasy Midi kit), and for the large scale (1 g maximum) (QIAGEN RNeasy Maxi kit). Unfortunately, currently available membrane based RNA extraction and purification systems require multiple steps, and may be too slow for intra-operative diagnostic applications.
Accordingly, a more rapid membrane-based RNA extraction method is needed particularly for use in intraoperative diagnostic applications.